Document 2q78vjzwK96wE1e3E2pj5deeN

Corning 14azietonInc. P.O. Box 7S45 Madison, Wi 53707-7545 Deliveries.3301 Kinsman Blvd. Madison, Wl S3704 608.241.4471 608.241.7227 Fax Sponsor: 3M St.Paul,Minnesota CORNINGHazleton FINAL REPORT Study Title: Single-DoseIntravenousPharmacokinetic Study ofT-6564 inRabbits Author: F.Bud W. McDonald Study CompletionDate: April18,1997 PerformingLaboratory: Coming HazletonInc. 3301 Kinsman Boulevard Madison,Wisconsin 53704 LaboratoryProjectIdentification: CHW 6329-186 Page I of34 Corning Pharmaceutical Services QUALITY ASSURANCE STATEMENT CHW 6329-186 Thisreporthas been reviewed by the QualityAssurance Unit of Coming Hazleton Inc.,in accordance with the Food and Drug Administration(FDA) Good Laboratory PracticeRegulations,21 CFR 58. The followinginspectionswere conducted and findingsreported to the Study Directorand management. InspectionDates From TO 06127196 09103196 10115196 12103196 06127196 09103196 10115196 12105196 Phase ProtocolReview Sample Collection ProtocolAmendment DatalReportReview Date Reported to Study Date to Director Management 06127196 09103196 10115196 12105196 06127196 09103196 10115196 12105196 Representative,QualityAssuranc@-tUnit Date 2 STUDY IDENTIFICATION Single-DoseIntravenouPsharmacokinetic StudyofT-6564 inRabbits CHW 6329-196 TestMaterial Sponsor Sponsor'sRepresentative Study Director Study Location Study Timetable Study InitiatiDoante Experimenta(lIn-life) StartDate In-lifEend Date ExperimentalTerminationDate Study CompletionDate T-6564 3M ToxicologyService MedicalDepartment 3M Center,Bldg.220-2E-02 P.O.Box 33220 St.Paul,NiN 55133-3220 Roger G. PerkinsP,hD, DABT 3M ToxicologyService MedicalDepartment 3M Center,Bldg.220-2E-02 P.O.Box 33220 St.Paul,NN 55133-3220 (612)733--)222 F.Bud W. McDonald Coming HazletonInc. P.O.Box 7545 Madison,Wl 53707-7545 (608)242-7901 Coming HazletonInc. '3301Kinsman Boulevard Madison,Wl 53704 August 2,1996 August 6,1996 September 3,1996 April18,1997 April18,1997 3 KEY PERSONNEL CHW 6329-186 Acute Studies QualityAssurance StevenM. Glaza Manager SherryR. W. Petsel Manager F.Bud W. McDonald Study Director LaboratoryAnimal Medicine JeffreyB. Hicks In-lifSeupervisor Rose M. Bridge AdministrativSeupervisor Cindy J.Cary,DVM DiplomateA,CLAM Supervisor AnatomicalPathology ToxicologySupport Kathy Myers Manager CalvinL. Horton Supervisor Thomas E. Palmer,PhD AnatomicalPathologist JackSerfort/ DeborahL. Pirkel Supervisors Necropsy Anne Mosher Supervisor PathologyData 4 CONNNTS QualityAssuranceStatement Study Identification Key Personnel Summary Objective RegulatoryCompliance Testand ControlMaterials Test System Procedures Results Discussion Signature Table I IndividuaBlody Weights(g) 2 IndividuaCllinicaSligns IndividuaAlnimal TissueWeightsand BileVolumes Appendix A ProtocolDeviations ProtocolTP6350 Amendment No. I totheProtocol CHW 6329-186 2 3 4 6 8 8 8 9 10 12 13 13 14 16 18 19 20 21 33 5 SLTMALARY CHW 6329-186 This study was done to assessthe levelof systemic exposure of T-6564 when administeredby a singleintravenousinjectionto rabbits. The study was conducted using fourmale and four female acclimatedrabbitsof the Hra:(NZW)SPF strainforeach treatmentgroup as follows: Group ControVTest Material Dose Level (mg/kg)@ I (Control) 2 Sterile Water 1 T-6564 T-6564 0.0 3.0 10.0 4 T-6564 100.0 5 T-6564 300.0 Number of AnimalSb Male Female 4 4 4 4 4 4 4 4 4c 4 a Administered ata dose volume of 0.5 mL/kg. b Two animals/sex/doselevelwere sacrificedon Day 14. The remaining animals (two animals/sex/doselevel)were sacrificeodn Day 29. c One male died approximately20 minutes aftertreatment(possiblydue to the volume and rateof injection)A. replacement animal was then selectedand treatedin the same manner. The animals receiveda singleintravenousinjectionof the controlor testmaterialatthe indicated dose levelintothe marginal ear vein of the rightear. The testmaterialwas mixed with Sterile Water forInjection(sterilweater)to a specificconcentrationforeach dose leveland administeredata dose volume of 0.5 mL/kg of body weight. One of the initiamlales treatedat ')00mg/kg (Group 5) died approximately20 minutes aftertreatment.This animal,discarded without an abbreviatednecropsy examinationor tissuecollectionw,as replacedwith another animal and treatedin the swne manner. Two animals/sex/doselevelwere sacrificedon Day 14 and theremaining animals (two animals/sex/doselevel)were sacrificeodn Day 29. Clinicalobservationswere conducted predose and atapproximately 0.5,2,and 4 hours after intravenousinjection.Additionalclinicaolbservationsand twice a day mortalitychecks were conducted dailythereafteurntilthescheduledsacrificeinterval(a.m.mortalitycheck only on days of scheduled sacrifice)B.ody weights were determined on Day -6 forrandomization 6 CHW 6329-186 purposesb,eforetestorcontromlaterialadministrati(oDnay 1),and atthescheduledsacrifice interva(lDay 14 or Day 29). A blood sample (approximately4 mL) was collectedfrom each animal fourdays before controlor testmaterialadministrationwith the exceptionof the one Group 5 replacement male which had a blood sample collectedon Day I (beforetestmaterial administration).Blood samples were alsocollectedfrom the animals at approximately4-,8-, 12-,24-,and 48-hours post-injectiona,nd on Day 8. An exceptionto theseintervalwsas the replacement animal (Group 5 male),from which blood samples were collectedatapproximately 3.5-,6.5-,and 10.5-hourspostdoseat the 4-,8-,and 12-hourpostdose intervalsr,espectively.An approximate 4-mL blood sample was alsocollectedon Days 14 and 22 from each animal scheduledforsacrificeon Day 29. In addition,atthetime of thescheduled sacrific(eDay 14 or Day 29),approximately20 to 40 mL of blood was obtainedfrom each controlanimal and approximately20 mL of blood was obtainedfrom each Group 2-5 animal with theexceptionof one Group 4 female on Day 14 from which only approximately 12 mL of blood was collected. All samples were centrifugedand separatesamples of serum and cellularfractionswere obtained and sentfrozen on dry iceto the Sponsor. On Day 14 or 29, the animals were anesthetizedwith sodium pentobarbitalb,led via theposteriorvena cava,and exsanguinated. An abbreviatedgross necropsy examination was not done, however, tissueswere collected.The whole liver,bile,and both kidneys from each animal were collectedw,eighed (volume only determined forbile),and sent frozento the Sponsor. Afterbeing intravenouslyinjectedwith T-6564 or the sterilweater control,allanimals appeared clinicallnyormal throughoutthestudy with theexceptionof theone Group 5 male ('300mg/kg) thatdied within20 minutes of dose administration.In addition,one Group 4 female (100 mg/kg) and one Group 5 female (300 mg/kg) exhibiteda slightlossin weight attheirrespective terminationinterval(Day 14). 7 OBJECTIVE CHW 6329-186 Theobjectiovfethisstudwyastoassestsheleveolfsystemeixcposurteothetesmtaterial, T-6564,when administeredas a singleintravenouisnjectiotnorabbits. REGULATORY COMIPLL4.NCE This studywas conductedinaccordancewiththeU.S. Food and Drug Administration'Gsood LaboratoryPracticeRegulationsforNonclinicaLlaboratoryStudies2,1 CFR 58,withthe exceptionthatanalysisof thetestmixturesforconcentratiohno,mogeneity/solubilitayn,d stabilitwyas not conducted.Allproceduresused inthisstudywere incompliancewiththe Animal WelfareAct Regulations.Intheopinionof theSponsorand studydirectort,hestudydid notunnecessarildyuplicateany previouswork. TEST AND CONTROL MATERIALS Identification The testmaterialwas identifieadsT-6564 and describedas a clearcolorleslsiquid.The control materialwas SterilWeater forInjectioUnS,P (AbbottLaboratoriesL,otNo. IO-J'82-JT; ExpirationNovember 1997),and was describedas a clearc,olorleslsiquid. Purityand Stability The Sppnsorassumes responsibiliftoyrtestmaterialpurityand stabilitdyeterminations (includinugndertestconditions)A.nalysisofthetestmateriamlixturesforconcentration, homogeneity/solubilitayn,d stabilitwyas notconductedorrequestedby theSponsor.The purity and stabilitoyf thecontrolmaterialwere consideredtobe adequateforthepurposesof thisstudy. Storageand Retention The testmaterialwas storedatroom temperature.The controlmaterialwas storedreftigerated. Reservesamples of thetestand controlmaterialswere takenand storedina freezersetto maintaina temperatureof-20*C 10*C. The controlmateriarleservesamplewillbe retainedat CHW forIyearin accordancewiththeWisconsinfacilitoyfComing Hazleton(CHW) Standard OperatingProcedure(SOP) and thendiscarded.The testmaterialreservesample and unused test materialwere returnedtotheSponsor.Any remainingcontrolmaterialmay be used forother testingand willnotbe discardedafterissuanceof thefinalreport. 8 CHW 6329-186 SafetPyrecautions ThetesatndcontromlaterihaalndlipnrgocedurweesreaccorditnogCHW SOPsandpolicies. TEST SYSTEM TestAnimal AdultalbinroabbiotfstheHra:(NZW)SPsFtraiwnerereceivferdomHR.PI,nc.K,alamazoo, Michiganon June 26,1996 and maintainedattheCHW facilitayt3')01Kinsman Boulevard, Madison, Wisconsin. Housina, Afterreceiptt,heanimalswere acclimatedfora periodof atleast7 days. During acclimationand throughoutthestudy,theanimalswere individuallhyoused insuspended stainlesssteelcagesin temperaturea-nd humidity-controlleqduartersE.nvironmentalcontrolfsortheanimalroom were settomaintaina temperatureof 19* to2'3*C,a relativheumidityof 50% *20%, and a 12-hourlight/12-houdrarklightincgycle.The darkcyclewas interruptetdoconductin-life procedures.In caseswhere variationfsrom theseconditionesxistedt,heywere documented and consideredtohave had no adverseeffecton thestudyoutcome. Animal Diet The animalswere providedaccesstowaterad libituamnd a measuredamount ofLaboratory RabbitDietHF -95)-2'6,PMI Feeds,Inc.The feedisroutinelaynalyzedby themanufacturerfor nutritioncaolmponents and environmentalcontaminants.Samples ofthewaterareperiodically analyzed.There were no known contaminantsinthefeedor wateratlevelsthatwould have interferewdith or affectedtheresultosfthestudy. SelectionofTest Animals The animalswere identifiebdy animalnumber and correspondingeartagand were placedinto studyaroupsbased on a stratifibeoddy weightrandomizationprogram. The randomizationbody %Veit_vwhetrse determinedon Day -6.A Group 5 replacementmale (AnimalNo. F60093) was arbitrarisleylectedfrom theextraanimalsacclimatedforthestudy. 9 CHW 6329-186 Study Design Animalsweighingfrom2,613to2,998g atinitiatioofntreatmenwtereplacedintothefollowing study groups: Group Control/Test Material Dose Level (mg/kg)@ Number of Animals' Male Female I (Control) Sterile Water 0.0 4 4 2 T-6564 3.0 4 4 3 T-6564 10.0 4 4 4 T-6564 1 100.0 1 4 4 5 T-6564 1 300.0 1 4c 4 a Administered ata dose volume of 0.5 mL/kg. b Two animals/sex/doselevelwere sacrificeodn Day 14. The remaining animals (two animals/sex/doselevel)were sacrificeodn Day 29. c One male died approximately20 minutes aftertreatment(possiblydue tothe volume and rateof injection)A. replacementanimal was then selectedand treatedinthe same manner. JustificationforSpecies Selection Historicallyt,he New Zealand White albinorabbithas been the animal of choice because of the largeamount of background informationon thisspecies. PROCEDURES Dose Preparation and Administration The testmaterialwas dilutedwith sterilweater to achieve a specificconcentrationforeach dose levelinGroups 2 to 5. An individualdose of each respectivecontrolmaterialor testsolution was calculatedforeach animal based on itsbody weight on the day of treatment.The control materialor respectivetestsolutionwas administeredby intravenousinjectionintothe marginal ear vein of the rightear over a"periodof 34 to 52 seconds. The prepared testsolutionswere storedat room temperatureuntiladministered.Afteradministrationa,ny remaining testsolutions were discarded. 10 ReasonforRouteofAdministration Intravenoiunsjectiiosannacceptabrloeutteoassesssystemeixcposure. CHW 6329-186 ObservatioonfsAnimals Clinicoablservatiwoenrseconductperdedosaendatapproximat0e.l5y2,,and4 hoursafter intravenouisnjectionA.dditionalclinicaolbservationasnd twicea day mortalitcyheckswere conducteddailythereafteurntilthescheduledsacrificienterva(la.m.mortalitcyheck onlyfor animalssacrificeodn Days 14 or29). Body weightswere determinedon Day -6 forrandomizationpurposes,beforetestorcontrol materialadministratio(nDay 1),and atthescheduledsacrificienterva(lDay 14 orDay 29). Blood Sample Collections/Shipment A bloodsample(approximatel4y mL) was collectefdrom a marginalearvein(lefetar)ofeach animalon Day -4,withtheexceptionof AnimalNo. F6009')(Group5 replacementmale)which had a blood sample collectefdrom theleftearon Day I (beforetestmaterialadministration). Blood samples(approximatel4y mL) werethencollectefdrom a marginalearvein(lefetar)of eachanimalatapproximately4-,g-,12-,24-,and 48-hourspost-injectioan,d on Day 8. An exceptiontotheseintervalwsas thereplacementanimal(Group 5 male),from which blood sampleswere collectedatapproximately3.5-,6.5-,and 10.5-hourspostdoseatthe4-,8-,and 12-hourpostdoseintervalsr,espectivelyA.n approximate4-mL bloodsample was alsocollected on Days 14 and 22 from each animalscheduledforsacrificoen Day 29. In additiona,tthetime ofthescheduledsacrific(eDay 14 orDay 29)viatheposteriovrenacava,approximately20 to 40 mL of blood was obtainedfrom eachcontrolanimaland approximatel2y0 ML ofblood was obtainedfrom each animalinGroups 2-5,withtheexceptionofAnimal No. F60085 (Group4 female)from which onlyapproximately12 mL of bloodwas obtained.Allsampleswere stored atroom temperatureuntilcentrifugedA.ftercentrifugatiosne,paratesamplesofserum and cellulafrractionwsere obtained.The senun and cellulafrractiosnamplesobtainedthrough Day 14 were storedina freezersettomaintaina temperatureof -20*C 10*C untilshipped frozen(on dry ice)totheSponsor(JamesD. Johnson,3M E.T.& S,Bldg.2-3E-09,935 Bush Avenue, St.Paul,MN, 55106),on Day 21. The serum and cellulafrractiosnamplesobtainedon Days 22 and 29 were storedatCHW and shippedtotheSponsorsixdays afterexperimental(inlifet)erminationinthesame manner asthesamplesobtainedpriortoDay 22. Pathology The Group 5 animal thatdiedshortlyafterdose administratiownas discardedwithoutan abbreviatednecropsyexaminationortissuecollectionO.n Day 14,thefirstwo animals/sex assignedtoeach dose level(basedon thegroupassignmentrandomizationw)ere anesthetized CHW 6329-186 withsodiumpentobarbi(tvaiilanjectinotnhemarginaelarvein)b,ledviatheposterivoerna cava,and exsanguinated.An abbreviategdrossnecropsyexaminationwas notdone,however, tissuewsere collected.The whole liverb,ile,and both kidneysfrom each animalwere collected, weighed (volume onlydeterminedforbile)a,nd immediatelyplacedina freezersettomaintaina temperatureof -20*C:EIO*C. Aftertissue/bicloellectiotnh,eanimalswere discarded.The remainingtwo animaWsex/dose levelwere anesthetizedb,led,and exsanguinatedon Day 29 in thesame manner astheanimalssacrificeodn Day 14. Shipment ofTissues The tissue(swhole liversand kidneys)and bilecollecteodn Days 14 and 29,alongwith documentationof theircorrespondinwgeightsorvolumes,were sentfrozen(ondry ice)tothe Sponsor(James D. Johnson,3M E.T.& S,Bldg.2-3E-09,9")5Bush Avenue, St.Paul,N4N, 55106) one week aftercollectioannd sixdaysafterin-lifteerminationr,espectivelyT.he Sponsorisresponsiblfeortheretentioannd dispositioonfthesamples.CHW does notaccept any responsibiliftoyrtheanalysisof thesamplescollecteidnthisstudynoraretheseresults presentedinthisreport. StatisticAanlalyses No statisticaanlalyseswere requiredby theprotocol. LocationofRaw Data,Records,and FinalReport The raw data,records,and an originaslignedcopy ofthefinalreportwillbe retainedinthe archivesof CHW inaccordancewithCHW SOP. RESULTS Body Weights Individuablody weightsareinTable 1. Allanimalsgainedweightduringthestudywiththe exceptionof one Group 4 female(100mglkg)and one Group 5 female(300mg/ka),sacrificeadt Day 14.which exhibitedweightlossesof 125 g and 13 g,respectively. ClinicalObservations IndividuacllinicaslignsareinTable 2. Allanimalsappearednormalthroughoutthestudywith theexceptionof theGroup 5 male (Animal No. F60071)thatdiedapproximatelytwentyminutes postdose.The deathof thisanimalisbelieveddue totherateand volume of theadministered dose. 12 CHW 6329-186 Pathology Individuaalnimaltissuweeightsand bilevolumesareinTable3. The animalswerenot examinedgrosslya,lthoughtissuewseresaved(withtheexceptionoftheGroup 5 malethatdied shortlyafterdosing -no tissueswere saved on thisanimal). DISCUSSION The levelof systemic exposure of T-6564 was evaluatedin male and female albinorabbitswhen administeredas a singleintravenousinjectionatlevelsof 3.0,10.0,100.0,and 300.0 mg/kg. All animals appeared clinicallnyormal throughout the study with the exceptionof the one Group 5 male (')00.0 mg/kg) thatdied within20 minutes of dose administration(believeddue to the rate and volume of thedose). All survivinganimals gained weight during thestudy with the exceptionof one Group 4 female (100 mg/kg) and one Group 5 female (')00 mg/kg) thatwere sacrificedatDay 14. SIGNATURE Q@F F. Bud W. McDonald Date Stud%,Director Acute Studies 13 T2ble 1 CHW 6329-196 IndMdual Body Weights (g) Animal Randomization Sex Number (Day -6) Initial (Day 1) Terminal (Day 14) (Day 29) Group I(Control)-SterilWeater (0.0mgfkg) Male Female F60052 F60053 F60054 F60055 F60072 F60073 F60074 F60075 2,643 2,752 2,653 2,591 2,828 2,774 2,797 2,777 2,724 2,788 2,686 2,685 2,904 2,981 2,805 2,980 2,919 2,884 - 2,914 3,008 - 2,833 2,907 3,207 3,229 Male Female F60056 F60057 F60058 F60059 F60076 F60077 F60078 F60079 Group 2 - T-6564 (3.0mgtkg) 2,898 2,798 2,716 2,821 2,923 2,698 2,699 2,882 2,962 2,851 2,790 2,94(l 2,924 2,873 2,799 2,950 Group 3 - T-6564 (10.0mg/kg) 3.012 2.937 - 3.122 3,141 - 3,024 3,149 3,082 3,242 Male Female F60060 F60061 F60062 F60063 F60090 F60081 F60082 F60083 2,738 2,634 2,716 2,832 2,732 2,520 2,769 2,786 2,822 2,667 2,912 2,749 2,797 2,613 2,838 2,795 2,924 2,822 - 2,923 2,818 - 3,293 2.984 3,147 3,108 Not required. Animal sacririceodn Day 14. 14 TableI (Continued) IndividuaBlody Weighb (g) CHW 6329-186 Aniff&W -Randominition Initial Terminal Sex Numba (Day-6) (Day1)- (Day14) (Day29) Group 4 -T-6564(100.m0gtkg) Male Female F60064 F60065 F60066 F60067 F60094 F60085 F60086 F60087 2,686 2,726 2,740 2,745 2,538 2,632 2,728 2,892 2,697 2,792 2,734 2,750 2,653 2,696 2,825 2,998 2,836 2,974 - 2,799 2,571 3.071 2,962 3,137 3,289 Group 5 -T-6564 (300.0mg/kg) Male F60068 F60069 F60070 F60071 F60093 b 2,774 2,590 2,917 2,633 2,989 2,865 2,722 2,936. 2,707 2,951 3,128 2,784 3,217 a 3,174 Female F60088 F60089 F60090 F60091 2,837 2,661 2,913 2,711 2,872 2,737 2,953 2,781 3,106 2,724 - 3,269 3.070 Not required. Animal sacrificeodn Day 14. Animal diedapproximatel2y0 minutespostdose. b ReplacementanimalforAnimal No. F60071. 15 Table 2 IndividuaCllinicaSligns CHW 6329-186 Sex Male Female Animal Number Observation Hour (Day Ir 0.5 2.0 4.0 Group I (Control)- SterileWater (0.0m&nW F60052 F60053 AppearedNormal AppearedNormal F60054 AppearedNormal F60055 AppearedNormal or F60072 F60073 F60074 F60075 AppearedNormal AppearedNormal AppearedNormal AppearedNormal Group 2 -T-6564(3.0mgtkg) Days 2-8 9-14 15-29 Male F60056 AppearedNormal F60057 AppearedNormal F60059 AppearedNormal F60059 AppearedNormal Female F60076 F60077 F60079 F60079 AppearedNormal AppearedNormal AppearedNormal AppearedNormal Group 3 - T-6564(10.0mgft) Male F60060 AppearedNormal F60061 AppearedNormal F60062 AppearedNormal F60063 AppearedNormal Female F60080 F60081 F60092 F60083 AppearedNormal Appewed Normal AppearedNormal AppearedNormal f Vol a Each animalappearednormalpriortocontrolortestmateriaaldministration. Animal sacririceodn Day 14. Conditionexisted. 16 Table2 (Continued) Individu2CilinicaSligns CHW 6329-186 Animal Sex Number Observation Hour(DayI)' 0.5 2.0 4.0 2-8 Group 4 -T-65& (100.0mg/kg) Male F60064 AppearedNormal v F60065 F60066 AppearedNormal AppearedNormal F60067 AppearedNormal Female F60094 F60085 F60086 F60087 AppearedNormal AppearedNormal AppearedNormal AppearedNormal Group 5 -T-6564(300.0mg/kg) Male F60068 AppearedNormal F60069 AppearedNormal F60070 AppearedNormal F60093 AppearedNormal Female F60088 F60089 F60090 F60091 AppearedNor7nal AppearedNormal AppearedNormal AppearedNormal a Each animalappearednormalpriortocontrolortestmateriaaldministration. Animal sacrificeodn Day 14. Conditionexisted. Days 9-14 15-29 17 TABLE 3 rndividual Animal Tissue Weights and Bile volumes Corning Hazleton Inc. Madison. Wisconsin USA --------------------------------------------------------------------------------------------------------------- TABLE INCLUDES: SEX-ALL;GROUP-ALL;WEEKS-ALL DEATH-A.LL,SUBSET-ALL ORGAN ABBREVIATION: Ll - LIVER, KD - KRDNEY, BI - BILE (ML) SEX DOSE ANIMAL SEX DOSE ANIMAL GROUP NUMBER - -Li- -(9-) - - K-D -(-9)- - -81- -(m-L) GROUP NUMBER - -L-i -(9-) - - K-D - (-9)- - -BI- -(m-L) ---------------------------------------------------- ---------------------------------------------------- Dose Level - 0.0 mg/kci N 1 P60052a 90.3302 14.6620 1.1000 p 1 P600720 79.9048 15.7815 0.8000 I F60053' 73.7863 16.7332 0.7000 p 1 F60073' 82.8800 14.6450 0.8000 00 N I F60OS4b p6ooSSb 48.1630 IS.2466 1.1000 F 1 P600740 71,9963 15.1540 0.7000 m I 76.1902 14.6761 1.0000 F I F60075" 92.6407 19.SO87 I.SOOO 9 2 P60OS6, m 2 P60057a m 2 F60OSgb m 2 F60DSgb 91.8360 60.80is 77.34S7 90.2474 15.161S 15.6406 17.9012 16.7929 Dome Level 0.3000 0.7000 0.2000 1.0000 3.0 mci/kci ;-- 2 F600764 F 2 P600770 F 2 F60078b F 2 F60079b 91.0291 lol.o6s9 80.7474 92.73S7 15.8879 IS.9660 13.9903 19.IGS7 0.4000 1.2000 1.1000 1.4000 m 3 F60060a m 3 P60061' m 3 F60062b m 3 F60063b 74.6403 75.0707 93.3989 68.3306 15.0898 16.0551 16.2391 1S.0642 Dose Level - 10.0 Mci/kq 0.7000 F 3 F60080a 0.1000 F 3 F60091a 1.6000 F 3 P60082b 0.6000 F 3 P60083b 86.6055 69.6640 99.7506 89.2276 16.3950 16.0235 18.3869 15.7710 1.6000 0.7000 0.8000 0.1000 m 4 * 4 * 4 * 4 F60064' P6006sa F60066b F60067b 63.9420 81.3660 74.4153 66.7900 15.24SO 15.6753 IS.4621 14.1424 Dose Level - 100.0 mg/ka 1.5000 F 4 P60004' 0.6000 0.9000 F 4 p 4 P6000s, F60096b 0,3000 F 4 F60087b 69.7363 00.6sis 88.9171 87.2201 14.4089 13.6620 13.2784 16.3357 0.8000 0.9000 1.0000 0.9000 m s F600686 m s F60069' m 5 F60070b m 5 P60093b 99.4912 01.8480 75.3822 80.5670 IS.2020 17.1618 16.5091 16.9704 Doag Level - 300.0 mg/kq iliooo p 5 F600884 0.9000 F 5 F600096 0.4000 0.9000 F 5 F60090b F s F60091b 91.9829 73.9520 91.9623 100.2084 1S.3994 12.3374 18.2069 17.9672 1.SODO 0.6000 2.0000 2.5000 a Sacrificed an Day 14. b Sacrificed on Day 29. APPENDIX A ProtocolDeviations Protocol'f?6350 Amendment No. I To'lbeProtocol CHW 6329-186 19 ProtocolDeviations CHW 6329-186 Protocol Page 8,7.ExperimentalDesign,D. ObservationofAnimals,(3)Blood Sample Collection(sb,) Method of Collection/Numberof Animals, Second Paragraph,Last Two Lines. Plapproximatel2y0 mL of bloodwill be obtainedfrom each Group 2-5 animalsacrificeodn Days 14 or29." Actual Procedure Only approximately12 mL of blood was collectefdrom one Group 4 female(AnimalNo. F60085) attheDay 14 sacrifice interval. Page 8,7.ExperimentalDesign,D. ObservationofAnimals,(3)Blood Sample Collection(sa,) Frequency, Second Line."administratitoon Day 1),4-,8-,12-,24-,and 48-hours post-". The bloodsamplescollectefdrom thereplacementanimal(AnimalNo. F60093) atthe4-,8-,and 12-hour postdoseintervalwsere collected 3-hours/21minutes,6-hours/41 minutes,and 10-hours/28minutes postdose,respectively. These deviationsarenotconsideredtohave had an adverseeffecton theoutcome ofthestudy. 20 Corning Haziaon Inc. P.O. Bas 7545 %Iadisoo.in 53707-7545 3301 gitsoun slid. Uiissnn. ri 531-04 60S.241.4471 60S.241.72-',-Fax Sponsor. 3M St Paul,Minnesota CHW 6329-186 CORNING Hazlcton PPOTOCOL TP6350 Study Title: Single-DoseIntravenousPhannacokinedcStudy of T-6564 inRAbbits Date: August2, 1906 PerformingLaboratory: CorningHazletonInc. 3301 Kinsman Boulevard Madison,Wisconsin 53704 LaboratoryProjectIdentification: CHW 6329-186 Corning PharrnaccuticaSlrviccs 21 C14W6329-186 STUDY MENTMCA77ON Single-Dose IntravenousPbarmacokineticStudy of T-6564 inRabbits CHW 6329-136 TP6350 Page 2 CHW No. TestMaterial Sponsor Sponsor's Representative Study Director Study Location Proposed Study Timetable ExperimentalSm Date ExperimentalTerminationDate DraftReport Date 6329-186 T-6564 3M ToxicologyService Medical Department 3M Center,Bldg.220-2E-02 P.O.Box 33220 SL Paul,MN 55133-3220 Roger G. Pakins,PhD, DABT 3M ToxicologyService Medical Department 3M Center,Bldg.220-2E-02 P.O. Box 33220 St.Paul,MN 55133-3220 (612)733-3222 F. Bud W. McDonald Coming HazletonInc. P.O. Box 7545 Madison,Wt 53707-7545 (608)242-7901 Coming HazletonInc. 3301 Kinsman Boulevard Madison,Wl 53704 August 6, 1996 September 3, 1996 October 15, 1996 1 22 CHW 6329-186 1. Study Singli.-DosIentravenouPsharmacokinctiSctudyin Rabbits CHW 6329-186 TP6350 Page 3 2. Purpose To assessthe levelof syst=ic exposurewhen thetea materialisadmwuered as a singleintravenouWsection torabbits 3. RegulatoryCompliance Thisstudywillbe conductedinaccordancewiththefollowingGood Laboratory PracticeRegulafions/Standards/Guidewhietshtheexceptionthatanalysisof the testmateriamlixturesforconcentratiosno,lubilithyo,mogeneity,and stabilitwyill not be conducted. ()Conduct asa NonregulatedStudy [)q21 CPR 58 (FDA) 40 CFR 160 (EPA-FIFPA) 40 CFR 792 (EPA-TSCA) C(81)30(Final()OECD) 59 NohSan No. 3850 (JapaneseMAFF) NotiricatiNoons.313 and 970 (JapanesMeOHW) AllproceduresinthisprotocoalreincompliancewiththeAnimal WelfareAct Regulations.In theopinionoftheSponsorand studydirectort,hestudydoesnot unnecessarildyuplicataeny previouswork. 4. QualityAssurance The protocols,tudyconduct,and thefinalreportwillbe auditedby the Quality AssuranceUnitinaccordancewiththeWisconsinfacilitoyf CorningHazleton Inc.(CHW) StandardOperatingProcedure(sSOPS)and policies. 5. TestMaterial A. Identification T-6564 B. PhysicalDescription (Tobe documentedintheraw data) 23 CHW 6329-196 CHW 632SLIS6 TP6350 Pap 4 C Purityand Stability The Sponsorassumesresponsibiliftoyrpm* and stabilidteyterminations (mcludingundertestconditions)S.amplesoftestmateri&YvehicmlLev=e(s) forconcentratiohno,mogeneity/solubilaintdys,tabili"tyy3es willnotbe takenbeforeadminisu-Aonunlessrequestedodmwise by theSponsor. These samplesCiftaken)willbe senttotheSponsoraftercq)crimental termination. D. Storage Room temperature E. Reserve Samples Reservesample(so)f eachbatch/lootftestmateriawlillbe takenforthis study. The testmateriarleservesample(s)willbe storedatCHW ina freezersetto maintaina temperaturoef -200C tIOeC and thenreturnedtotheSponsor aftercompletionof thein-lifpehaseofthestudy. F. Retention Any unusedtestmateriawlillbe returnetdotheSponsoraftercompletionof allrelateidn-lifteesting. G. SafetyPrecautions As requiredby CHW SOPs and policies 6. ControlMaterial A. ldentirication SterilWeater forInjectio(nsteriwlaeter) B. PhysicalDescription Clearcolorleslsiquid C. Purityand Stability The purityand stabilitoyfthisUSP grademateriailsconsideredadequatefor thepurposesof thisstudy. D. Storage Refrigerated 24 CHW 6329-186 CHW 6329-186 TP6350 Page 5 & Reserve Samples Reservesample(so)f eachbatchaotof contromlateriawlillbe takenforthis study. The contromlateriarleservesample(sw)illbe storedatCHW ina freezesret tornaintaiantemperatureof-200C tiOOC. F. Retention Any remainingcontrolmateriamlay be usedforothertestinagnd willnotbe discardedafterissuanceof thefinalrepom G. SafetyPrer-autions As requiredby CHW SOPs and policies 7. ExperimentalDesign A. Animals (1) species Rabbit (2) Strain/Source Hn:(NZW)SPFIIW. Inc, (3) Ageat Initiation Adult (4) Weight at Initiation 2.5to3.5 kg (5) Number and Sex 20 males and 20 females (6) Identification Individuanlumbered eartag (7) Husbandry (a) Housing Individualliyn,suspendedscrew-bottomstainlessteelC-ages 25 CHW 6329-186 CHW 6329-186 TP6350 Page 6 (b) Food A measuredamount ofLabonttorRyabbitDietBF #5326 (Phg Feeds,lnr-).The foodisroutinelaynalyzebdy theu=ufactu= fornutritioncaolmponentsand environmentaolonuminants. (c) Witter Ad libitufmroman automatiscystem.Samplesofthewaterare analyzedfortotaldissolvesdolidss,pecifiemdicrobiological contenkselectedelements,heavy metals,organophosphatesa,nd chlorinatehdydrocarbons. (d) Contaminants Thereareno known contaminantisnthe foodorwaterthat would interferweiththisstudy. (e) Environment Environmentaclontrolfsortheanimalroom willbe setto maintaina temperaturoef IrC to23*C,a relativheumidityof 501/a200/9a,nd a 12-hourlight/12-hoduarrkcycle.The dark cyclemay be interrupteddue toin-lifperocedures. (f)Acclimation At least7 days (8) Selectioonr TestAnimals Based on healthand body weightaccordingtoCHW SOPS. An adequatenumber ofextraanimalswillbe purchasedso thatno animal inobviouslypoorhealthisplacedon test.The animalswillbe placed intostudygroupsusinga stratifibeoddy weightrandomization programwithinninedaysof studyinitiation. (9) JustificatifoonrSpeciesSelection Historicalltyh,eNew ZealandWhite albinorabbithasbeen the aninud ofchoicebecauseof thelargeamount ofbackgroundinformatioonn thisspecies. 26 CHW 6329-186 B. Dose Administration (1) TestGroups CHW 6329-186 TP6350 Page 7 ControVrestDwe Level NumberofAnima Group material (m&W Male Female I Sterile (Control) Water 0.0 4 4 2 T-6564 3.0 4 4 3 T-6564 10.0 4 4 4 T-6564 100.0 4 4 5 T-6564 300.0 4 4 a Administeredata dosevolume of 0.5mLJkg. b Two animaLVscyJdosleevelwillbe sacrificeodn Day 14. The remaininganimals(two animalstsextdolseevelw)illbe sacrificeodn Day 29. C DosingProcedures (1) Dosing Route Intravenouisnjectioinntothemarginalearvein of therightcarover approximately30 to60 seconds. (2) Re2son forDosing Route Intravenousinjectioinsan acceptableroutetoassesssystemic exposure. (3) DosingDuration Singledose (4) Dose Preparation The day of treatmenwtillbe designatedasDay 1. Individuadloses willbe calculatebdasedon theanimal'sbody weighttakenon Day 1. The Group I animalswillbe treatedwithsterilweaterata dose volume of0.5mukg. The testmateriawlillbe dilutewdithsterile watertoachievea specificoncentratiofnoreach'doselevelin Groups 2-5 and administereadta dosevolume of0.5mLAg. The preparedtestmixtureswillbe storedatroom temperaturuentil administered. 27 CHW 6329-186 D. ObservationofAnimals CHW 6329-196 TP6350 Pap 8 (1) ClinicaOlbservations Beforetestor contmlmateriaaldminl@on, atapproximatelOy.S. 2.0,and 4.0hourspost-injwdo(nDay 1)forclinicasligns,daily themfterforclinicsailM andtwicedaily(LaL and pm) for mortalituyntilthescheduledsacrificienterva(lDay 14or Day 29). The anirnalsacrificeodn Days 14 and 29 willbe observedonlyonce formortalitoyn thoserespectivdeays. Observationmsay be extended when directebdy thestudydirector. (2) Body Weights Forrandomizatiobne,foretestor contromlateriailnjectio(nDay 1).at thescheduledsacrificienterva(lDay 14 or Day 29),oratunscheduled deathand sacrific(ewshen survivaelxceedsI day) (3) Blood Sample Collections (a) Frequency Pre-injecti(oannytimefrom up tofourdaysbeforetestmaterial administratitoonDay 1),4-.g,-,12-,24-,and 49-hour3postinjectiono,n Days 8,14,22@and atthescheduledsacrifice interva(lDay 14 orDay 29) (b)Method ofCollection/NumbeorfAnimals Blood samples(approximatel4ymL) willbe collectefdrom the marginalem vein(titheerar)of allanimalson thedaybefore testorcontromlateriailnjectioanndfrom themarginalcarvein (lefctar)at4-hourspostdosethroughDay S. On Days 14 and 22,additionabloodsamples(approximatel4ymL) willbe wilectedfrom themarginalearvein(lefctar)oftheanimals scheduledforsacrificoen Day 29. From theposteriovrenacava,approximatel2y0 mL of blood willbe obtainedfrom eachanimalsacrificeidna moribund conditio(nifpossiblea)p,proximatel2y0 to40 mL ofbloodwill be obtainedfrom eachcontrolanimalsacrificeodn Days 14 or 29 (themaximum volumepossiblweillbe obtained)a,nd approximatel2y0 mL ofbloodwillbe obtainedfrom each Group 2-5 animalsacrificeodn Days 14or 29. 28 CHW 6329-186 CHW 6329-196 TP6350 Page 9 The sampleswillbe storedatroom t=pcmture and then ctmuffugeda,nd theseparatteen= and cellulafrractionsstored ina free= settomaintaina temperaturoef -20*C:tlD*C.The senun and cellulafrractionosbtainedthroughDay 14wiU be sentfrozenon dry icetotheSponsorone totwo weeks priorto in-liftermination7.te serumand cellulafrractionosbtained afterDay 14 willbe sentfrozenon dryicetothe Sponsorwithin one week afterin-lifteerminationT.he Sponsorisresponsible fortheretentioannd dispositioofnthes=ples. The serum and cellulafrractiosnampleswillbe shippedto: James D. Johnson 3M E.T.& S Bldg.2-3E-09 935 Bush Avenue SL Paul,MN 55106 James D. Johnsonor hisalternatweillbe notifierdegardingthe shipmentof thesamples. L Termination (1) UnscheduledSacririceasnd Deaths Any animaldyingduringthestudyorsaciiriceidna moribund conditionwillbe subjectedtoan abbreviategdrossnecropsy examinationandallabnormalitiweisllbe recorded.Animals ina moribund conditiownillbe anesthetizweidthsodium pentobarbit(avlia injectioinnthemarginalcarvein)b,ledviathevena eava,and exsanguinatedT.issuesa,s describeidnsection7.E.(3)Sample Collectiowni,llbe collectefdrom any animaldyingduringthestudyor sar-riricienda moribundconditionA.fternecropsyt,heanimalswill be discarded. (2) ScheduledStcrirtces On Day 14,therirstwo animals/seaxssignedtoeachdose level (basedon thegroupassignmentmndomization)willbe anesthetized withsodium pentobarbit(avliainjectioinnthemarginalcarvein),bled viathevenacava,and cmmguinated. The rema7inintgwo animalstsex/doslevelwillbe anesthetizewdithsodium pentobarbital (viainjectioinnthemarginalcarvein),bledviathevena cava,and exsanguinateodn Day 29. An abbreviategdrossnecmpsy examination 29 CHW 6329-196 CHW 6329-196 TP6350 Page 10 willnot be done.h,owever,tissue(sasdocnbed insection7.E.(3) Sample Collectiowni)llbe collected. (3) Sample Collection The whole liverb,ile,andbothkidneys(collecteadsonesample)fi-om eachanimalwillbe collectewde,ighed(volumeonlydeterminedfor bile)a,nd immediatelyplacedintoa freezersettomaintaina temperaturoef-20*CIOIC. Aftersamplecollectiotnh,eanimalswill be discarded. The samples(liverb,ile,andkidneys)willbe sentfrozenon dry iceto theSponsorwithinone week aftercollectionT.he samplesand their correspondinwgeightsorvolumes willbe shippedtothepersonlisted inSecion 7.D.(3)(b)T.he Sponsorisresponsibfloertheretentioannd dispositioonfthesamples. F. StatisticAanlalyses No statisticaanlalysesarerequired. S. Report A rinalreportincludintghoseitemsWed below willbe submitted. Descriptioonf thetestand controlmaterials Descriptioonfthe testsystem Procedures Datesofexperimentailnitiatiaonnd termination Descriptioonf any toxiceffects Gross pathologyrinding(sifapplirable) Grosspathologyreport(ifapplicablaend requestedby thestudydirector) Individuaalnimaltissuweeightsand bilevolumes 30 C14W 6329-196 CHW 6329-186 TP63SO Page 11 9. Location of Raw DaM Resove SamPit(s),Records, and FtaalReport Originaldata,or copiesthereofw,illbe availablaetCHW to facilitaatuediting thestudyduringitsprogressand beforeacceptanceof thefinalreport When the finalreportiscomplet4 controlmateriarleserves=ple(s),alloriginaplaper data,includingthoseitemslistebdelow willbe retainedin thearchivesof CHW fora periodof otleyearfollowingsigmng of thefinalreport.One yearaft= signingof thefinalreport,allof theaforementionedmaterialwsillbe sentto the Sponsor and a returnfeewillbe charged.The Sponsor may electto have the materialsretainedin theCHW Archivesforan additionapleriodof time and Ch'W willchargea storagefee. IftheSponsorchoosesto have CHW disposeof thematerialsa, disposalfeewillbe charged. Protocoland protocolamendments Dose preparationrecords In-lifreecords Body weights Dose administration Observations Sample collectiornecords Shippingrecords Pathology FLecords Study correspondence Finalreport(originaslignedcopy) The followingsupportingrecordswillbe retaineadt CHW but willnotbe a.rchivewdith the studydata. Animal receipt/acclimatiroencords Water analysisrecords Animal room temperatureand humidityrecords P,efrigeratoarnd freezertemperaturerecords Instrumentcalibratioannd maintenancerecords 31 CHW 6329-196 PROTOCOL APPROVAL CHW 6329-186 TP6350 Page 12 .ASIJAK?IA LIA' Roger G71--l@nsP,hD, DABT Date Sponsor'sRepresentative 3M 4J F.Bud W. McDorWd Bate StudyDirector Acute Studies Coming HazletonInc. Representative Date QualityAssuranceUnit Coming HazletonInc. (6329-186)HD 32 4$ CHW 6329-186 Deiigv"m: JJOI Kim~ maii@. ri fAll.24 PAX Bti-J. 51704 CORNING Hazicton AMENDMENT NO. ITO THE PROTOCOL PROTOCOL TP6350 Single-DoseintravenouPsham=okinedc Studyof T-6564 in Rabbits CHW 6329-186 Sponsor TestingFacility 3m ToxicologyService MedicalDepartment 3M Center.Bldg.2-')-02E-02 P.O.Box 33220 St.Paul.MtN 55 133-3220 Coming HazletonInc. 3301 Kinsman Boulevard Madison.Wl 53704 Sponsor'sRepresentative Study Director Roger G. Perkins.PhD F.Bud W. McDonald This amendment modiricsthe follo%%ipnogrtioonftheprotocol: EffectivAeu:ust6,1996 I Pa-.c7,7.ExperimentalDesi:n,C.DosingPrectdurts.Add thefollowinsgectionto theprotocol: (5) DieodnTest/ReplacAneimmeanlst Animal No. F60071 (Group 5 male) diedapproximatelyt%ventyminutes after treatment.Discard the animal withoutin abbreviatedgross necropsy examination or tissuecollection.Arbitrarilsyelecta replacementanimal (maic).weighing from 2.5 to 3.5 kg. from the cctm animals and obtaina predose (Day 1) 4-mL blood samplc. Process the sample according tothe procedure used forDa'! -4 samples. Treatthe replacementanimal on Day I inthesame manner used for the initiaalnimal and follow allsubsequentstudy proceduresas indicatedin the protocol. 33 CHW 6329-186 Amendment No. I CHW 6329-186 Page 2 Reason forabove change: To addresstheproceduresassociate-dwiththedeathand replacementofthenWe treateadt300.0mgtkg of body weight(Gmup 5). PROTOCOL AMENDMENT APPROVAL QRooerrP erkins Sponsor'sRepresentative 3M F.Bud W. McDonald StudyDirector AcuteStudies Coming HazletonInc. 14"Z /@ -y4, RepresAtative QualityAssuranceUnit Coming HazletonInc. (6329-186.Aml/HD) 10 1;2-91CL Date' 4v. zs t9,F& Date Date 34